Phospho c myc

Western Blotting analysis was performed as previously described^{38 (link)} in AGS and KATO III cells. Primary antibodies were directed against the following proteins: VEGFA and beta III Tubulin (abcam, Cambridge, UK), VEGF Receptor 2, Phospho-Tuberin/TSC2 (Ser939), Tuberin/TSC2, Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199), PI3 Kinase p110α, Phospho-Akt (Thr308), Akt (pan), Phospho-S6 Ribosomal Protein (Ser235/236), S6 Ribosomal Protein, 4E-BP1, Phospho-MEK1/2 (Ser217/221), MEK1/2, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), Phospho-SAPK/JNK (Thr183/Tyr185), JNK2, Phospho-c-Jun (Ser63), c-Jun, E-Cadherin, N-Cadherin, cdc25A, Phospho-cdc2 (Thr161), cdc2, Cyclin B1, Phospho-c-Myc (Ser62), c-Myc, Phospho-Stat3 (Ser727), Stat3, Phospho-Histone H2AX (Ser139) and β-actin (Cell Signaling, Beverly, MA, USA). The protein expression was quantified using the ImageJ software (http://rsb.info.nih.gov/ij/).

Drug-treated OECM-1 and SG cells were lysed in RIPA buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, and 1% Triton 100) at 4 °C. Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), α-tubulin, acetylated-α-tubulin, DRP1, GFP, VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-Myc/N-Myc, phospho-c-Myc (Ser62), phospho-c-Myc (Thr58), p-DRP1, HIF-1α (Cell Signaling, Danvers, MA, USA), cyclin D1 and p21 (Abcam, Cambridge, UK).

Human RANKL was obtained from Peprotech (Rocky Hill, NJ, USA). Antibodies (1:1000) used for immunoblotting are as follows: NFATc1 (Santa Cruz, Dallas, TX, USA; 7A6); c-Myc and Lamin B (Abcam; ab32072 and ab16048); a-tubulin (Sigma-Aldrich; T9026); Phospho-c-Myc, NRF2, ERK1/2, p-ERK1/2, IκBα, p-JNK and p-p38 (Cell Signaling Technology, Danvers, MA, USA; 13748, 12721, 9102, 9101, 9242, 9251, and 9215). CDDO-Im, U0126, SP600125, Ly294002, and SB203580 were purchased from Tocris (Minneapolis, MN, USA; 4737, 1144, 1496, 1130, and 1202). N-acetyl-l-cysteine (NAC) and actinomycin D were purchased from Sigma-Aldrich (A9165 and A1410).